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Regulation of apoptosis in aquatic organisms by aquabirnavirus

a technology of aquabirnavirus and apoptosis, which is applied in the direction of biocide, peptide/protein ingredients, antibody medical ingredients, etc., can solve the problems of not providing any insights which delineate the apoptosis process, and achieve the rescue or delay of the apoptosis cell death process, prevent the induction of dna, and maintain the expression level of mcl-1

Inactive Publication Date: 2006-03-07
ACAD SINIC
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  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0022]This method also applies to in vitro transfection of IPNV into fish cell lines, such as CHSE-214 (an embryo cell line from salmon). The preferred temperature for infected cultures is at 18° C. The induction of apoptosis by aquabirnavirus can be prevented by pretreatment of cyclohexamide (protein synthesis inhibitor), genistein (tyrosine kinase inhibitor), and EDTA (cation chelator) prior to viral infection.
[0025]The third method for inducing apoptosis include down regulating Mcl-1 gene expression. The down regulation of Mcl-1 gene expression correlates to IPNV replication. The preferred aquatic organisms are fish, in particular fish cells derived from salmon, trout, grouper, and eel. The down regulation of Mcl-1 gene expression can be blocked by drugs such as cyclohexamide (protein synthesis inhibitor), genistein (tyrosine kinase inhibitor), and EDTA (cation chelator). These drugs help to maintain Mcl-1 expression level and block the induction of DNA internucleosomal cleavage (i.e., blocking the intense DNA laddering pattern), so as to rescue or delay the apoptotic cell death process.

Problems solved by technology

's report did not provide any insights which delineate the apoptotic process from necrosis.

Method used

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  • Regulation of apoptosis in aquatic organisms by aquabirnavirus
  • Regulation of apoptosis in aquatic organisms by aquabirnavirus
  • Regulation of apoptosis in aquatic organisms by aquabirnavirus

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embodiment 1

orphological Changes During Apoptosis

[0061]Green fluorescent protein (GFP) from the jellyfish Aequorea victoria is a revolutionary report molecule for monitoring gene expression and fusion protein localization in vivo or in situ and in real time. One of the most useful aspects of GFP for biological studies is that it can be monitored in living cells. In the present study, a variant type of GFP (EGFP) serves as a marker for visualizing the dynamic apoptotic cell morphological changes and for tracing membrane integrity changes during apoptosis by fluorescence microscopy, as well as for quantitation of the intra- and extracellular release of EGFP during apoptosis by western blotting and fluorometry.

[0062]The use of EGFP to study the apoptotic process is illustrated in the following experimental designs, results, and discussion:

(A) Experimental Designs

(1) Wild-Type CHSE-214 Cells, CHSE-214-EGFP Cells, and Viruses

[0063]Chinook salmon embryo cells (CHSE-214) were obtained from American Ty...

embodiment 2

ell Death Via An Mcl-1 Dependent Pathway

[0084]Mcl-1 belongs to the Bcl-2 family, which is known as the “apoptosis-inhibiting protein”. The founding member of this family is the bcl-2 protooncogene which was initially isolated from a follicular lymphoma (Bakhshi et al. (1985), Cell 41:889–906). Mcl-1 was originally identified from the differentiating human myeloid leukemia cell line ML-1. Its expression was found to increase early in the induction or “programming” of differentiation of ML-1 cells before the appearance of differentiation markers. The coding region of mcl-1 was sequenced and found to have a pronounced region of sequence homology to bcl-2 in the carboxyl-terminal region (Kozopas et al. (1993), supra). Unlike bcl-2, mcl-1 contains a strong PEST sequence (enriched in proline, glutamic acid, serine and threonine) which is present in a variety of proteins that undergo rapid turnover. Overexpression of exogenously introduced mcl-1 has been shown to cause a prolongation of vi...

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Abstract

The present invention provides a mechanism for studies of apoptosis in aquatic organisms by infecting the aquatic organisms with aquabirnavirus, especially infectious pancreatic necrosis virus (IPNV). The infection of IPNV in an aquatic cell such as a Chinook salmon embryo cell (CHSE-214) converts the cell into an apoptotic cell. The present invention also provides a method for monitoring the morphological changes during apoptosis by cloning EGFP (a variant type of GFP) to an aquatic cell and monitoring the fluorescence using microscopic techniques. The intensity of the fluorescence reflects the expression of EGFP in cells. Finally, the present invention provides means for inducing or preventing / rescuing apoptosis in a host, which include aquatic and vertebrate. The apoptosis can be induced by IPNV infection or VP3 transfection that VP3 is a 32 kDa protein derived from IPNV segment A. The apoptosis can be prevented or rescued by an antisense VP3 RNA or a zfMcl-1a protein.

Description

RELATED INVENTION[0001]This patent application is a divisional of U.S. patent application Ser. No. 10 / 080,656, filed on Feb. 25, 2002 now U.S. Pat. No. 6,630,456, which in turn is a CIP of U.S. patent application Ser. No. 09 / 706,869, filed on Nov. 7, 2000 (now abandoned), which in turn claims the priority of U.S. Provisional Application Ser. No. 60 / 167,010, filed on Nov. 23, 1999, which are incorporated herein by reference.FIELD OF THE INVENTION[0002]The present invention, certain aspects of which are published in Virus Research (1999), 63:75–83 and J. Virol. (1999), 73:5056–5063 (which are herein incorporated by reference), relates to apoptosis in aquatic organisms induced by aquabirnavirus, preferably infectious pancreatic necrosis virus (IPNV). It also relates to methods to regulate apoptosis in aquatic cells by controlling the expression of Mcl-1 gene and pre-treating the aquatic cells with drugs which block the viral replication. It further relates to a method for monitoring th...

Claims

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Application Information

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IPC IPC(8): A01N55/00A01K61/00C12N7/00C12N9/00A61K38/16C12Q1/68
CPCC12Q1/6897A61K38/162
Inventor WU, JEN-LEIHHONG, JIANN-RUEY
Owner ACAD SINIC
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